As an ongoing project, the biochemical and molecular characteristics of thiamine-dependent enzymes involved in glucose metabolism were studied. These enzymes are mitochondrial pyruvate dehydrogenase (PDH) complex and `-ketoglutarate dehydrogenase (`-KGDH) complex, and cytosolic transketolase. These enzymes from bovine and rat tissues were purified to apparent homogeneity on SDS polyacrylamide gel electrophoresis and used for biochemical characterizations. A hydrophobic fluorescent probe, bis- ANS, potently inhibited the activity of PDH complex and `-KGDH complex. The conformational changes in these enzymes were demonstrative upon the addition of allosteric regulators such as ATP or ADP. In addition, PDH phosphatase was purified using PDH E2 affinity column chromatography. This purified enzyme was more sensitively inhibited by several antipsychotic drugs calmodulin antagonists via non-competitive manner with a following potency order: fluphenazine greater than chlorpromazine greater than thioridazine greater than perphenzine greater than triflupromazine greater than promazine. However, the activity of PDH complex was little or minimally affected. Near full-length cytosolic transketolase was cloned and its nucleotide sequence was determined. Liver-specific activation of transketolase was demonstrated using cloned cDNA probe and polyclonal anti-bodies. Based on the N-terminal amino acid sequences of the purified proteins, cDNA clones for mitochondrial PDH phosphatase, NADP+-specific, and NAD+-specific isocitrate dehydrogenases were also identified and characterized.